The term “peptide aptamer” is a general term for an artificial peptide that binds specifically to a specific target molecule. At present, small peptide aptamers that exhibit binding functions similar to “antibodies” have drawn much attention as probes for molecular detection, inhibitors of a biological functions, or the like in both chemical research and biological science research. Further, in the field of medicine, they are also expected to serve as molecular target drugs for the next generation in place of antibody pharmaceuticals.
Recently, a phage display method has been mainly used as a technique of creating peptide aptamers. In this technique, a strategy employed is to select a peptide aptamer that specifically binds to a target molecule from about 109 kinds of peptide libraries that are displayed on part of a coat protein of a phage. Yet, there are a number of problems remained to be solved. For instance, (1) in a process of selecting the peptide aptamer, the life cycle of Escherichia coli and phages is utilized and therefore peptides that adversely affect their life activity end up being automatically eliminated. Hence, a phenomenon that no peptide aptamers having an intended function are obtained often takes place. Further, (2) there is bias in the occurrence frequency of each of the codons coding for 20 kinds of amino acids in cells, and there is concern in that a large gap is created between the variety of theoretically designed-synthesized peptide libraries and that of libraries actually used. Furthermore, (3) what frequently happens to a peptide aptamer selected by this technique is a phenomenon that the properties to bind to a target molecule end up decreasing or disappearing in a state where it is separated from the coat protein of phage. That's because a state where the peptide aptamer and the protein derived from the phage are fused is essential for developing and maintaining the binding property to the target molecule. In the case of using the phage display method, this is an unavoidable serious issue. Hence, in order to avoid the above problem, it is required to construct a peptide library or protein library by not using living cells but using only intracellular translation reactions. And, development of an “in vitro display method”, by which a peptide aptamer specifically binding to an intended target molecule can be efficiently selected from a library, is imperative.
As such an in vitro display method, there has been a ribosome display method (Patent documents 1 to 4 and Non-patent document 1). As compared with a mRNA display method, in the ribosome display method, peptide (protein) libraries of various sizes can be designed and utilized in accordance with research applications and furthermore, an intended peptide aptamer can be selected and identified from those libraries by a quick and simple process. Therefore, the research can evolve with a view to commissioning and supplying in a kit or automation by robots in the future. However, because a peptide-ribosome-mRNA complex peptide aptamer that is used in the selection process is very unstable, it often happens that the intended peptide aptamer can not be identified. Therefore, even throughout the world, very few researchers are capable of freely dealing with this principle, condition, and technique in the present situation.
In order to increase the stability of the peptide-ribosome-mRNA complex, Non-patent document 2 discloses a technique in which Cv sequences are incorporated into the 5′-untranslated region of mRNA and Cvap dimer is included in a polypeptide to be expressed to carry out a ribosome display (Non-patent document 2). Yet, in order to increase the efficiency of the ribosome display, further improvement has been demanded.